The increasing incidence of obesity is a major health issue facing the United States today. An increased understanding of body weight regulation is critical in the prevention of obesity. Leptin is esablished as a critical hormone in the regulation of food intake, endocrine status and autonomic function, but the CNS pathways mediating leptin action are not fully defined. However, it is now established that leptin exerts many of its effects by acting through central melanocortin pathways and neurons expressing melanocortin-4 receptors (MC4-Rs). The identified MC4-R agonist is alpha-melanocyte stimulating hormone (MSH), a product of pro-opiomelancortin (POMC). Uniquely, an endogenous MC4-R antagonist exists, agouti-related protein (AgRP) that is expressed with neuropeptide Y (NPY) in neurons in the arcuate nucleus of the hypothalamus (Arc). In the preceding grant period, we identified several candidate pathways through which leptin may exert its effects. In this proposal we offer a model detailing the actions of leptin to regulate endocrine, autonomic, and behavioral responses. Specifically we propose that leptin differentially acts on POMC and AgRP neurons that innervate key neurons in the hypothalamus, brainstem, and spinal cord expressing MC4-Rs. We offer a series of neuroanatomical, electrophysiological, and genetic experiments to test components of this model. Using retrograde tract tracing and in situ hybridization, we will identify the chemical identity of CNS neurons expressing MC4-R and we will determine if neurons expressing MC4-Rs innervate key CNS autonomic control sites. Next, by combining neuroanatomical techniques with electrophysiology, we will assess the responses to leptin of POMC and AgRP neurons that innervate the lateral hypothalamic area, the paraventricular hypothalamic nucleus, and autonomic preganglionic neurons. Finally, using genetically modified mice we will assess the physiological role of MC4-Rs expressed in autonomic preganglionic neurons in the brainstem and spinal cord. We will use a mouse model containing a lox-modified, null allele of the MC4-R gene. The null allele is designed such that it contains p-lox sites flanking a transcription termination cassette so that expression of the MC4-R can be reactivated by expression of Crerecombinase. We propose to reactivate expression of MC4-R in autonomic preganglionic neurons by crossing the mice with transgenic mice that express Cre-recombinase under the control of the choline acetyl transferase (Chat) promoter. Thus, we will produce mice with MC4-R expression only in cholinergic neurons, including autonomic preganglionic neurons.